Alzheimer's Disease (AD) represents one of the great unsolved medical needs confronting society during this millennium. Despite considerable work during the past quarter century, no medicines exist that attack the underlying pathophysiology of the disease. One of the cardinal features of AD is deposition of plaques comprised of aggregated beta-amyloid peptides (Aβ) in the brain, particularly in regions associated with cognition and memory. Selkoe, Annu. Rev. Neurosci., 17, 489–517 (1994). Overproduction of Aβ, which appears to be directly neurotoxic, can be detected at the earliest stages of AD and, in fact, before cognitive dysfunction is detectable. Aβ is produced from its precursor protein, APP, by proteolytic processing at its N and C termini by β-and γ-secretase enzymes, respectively. Mutations in APP, presenilin-1, or presenilin-2 genes result in over-production of Aβ1-42 peptide and cause early onset, familial AD. The identity of the β-and γ-secretases have been studied since 1984, and in 1999 the elusive N-terminal β-site APP cleaving enzyme (BACE-1) was reported. Yan, et al., Nature, 402, 533–537 (1999). It remains possible that there are additional proteases with β-secretase activity.
BACE-1 mRNA is expressed widely at low levels, in the brain at moderate levels and in the pancreas at higher levels. However, β-secretase activity is low in pancreas and highest in brain. This discrepancy between mRNA and activity is probably due to a splice variant lacking two-thirds of exon 3 being the predominant BACE-1 transcript in pancreas, resulting in a protein that is incompletely processed and retained in the endoplasmic reticulum. In the brain, BACE-1 mRNA is widely expressed only in neurons, with most pronounced expression in the cerebellum, cortex, and hippocampus observed by in situ hybridization. BACE-1 is co-localized with its substrate, APP, in the trans-Golgi network of cells.
Because small-molecule BACE-1 inhibitors are being studied as potential Alzheimer's disease pharmacotherapeutics, it is necessary to prove that BACE-1 is the primary β-secretase of brain and to understand what effects BACE-1 inhibitors may have beyond inhibition of APP processing. It is also important to show that BACE-1 comprises the major β-secretase activity in brain. This disclosure reports and claims those findings and inter alia their use in methods of screening.